Crystal structure of the ITK kinase domain

Abstract

Disclosed are polypeptides encoding the ITK kinase domain and nucleic acids encoding such polypeptides, crystal structures of various polypeptide-ligand complexes comprising the ITK kinase domain bound to a ligand, methods of producing the aforementioned polypeptides and nucleic acids which encode them and methods of producing crystal structures of the aforementioned polypeptides comprising the ITK kinase domain bound to a ligand.

1 . A crystalline composition comprising an ITK kinase domain-ligand complex.  
     
     
         2 . The crystalline composition according to  claim 1  wherein the ITK kinase domain is human.  
     
     
         3 . The crystalline composition according to  claim 2 , wherein said composition effectively diffracts X-rays such that the atomic coordinates of the ITK kinase domain-ligand complex can be determined to a resolution of better than 5.0 Angstroms.  
     
     
         4 . The crystalline composition according to  claim 2 , wherein said composition effectively diffracts X-rays such that the atomic coordinates of the ITK kinase domain-ligand complex can be determined to a resolution of 3.0 Angstroms or better.  
     
     
         5 . The crystalline composition according to claims  2 ,  3  or  4  wherein the ITK kinase domain is chosen from ITK/KD/Q343 (SEQ ID NO. 9), ITK/KD/G354 (SEQ ID NO. 1) and ITK/KD/S361 (SEQ ID NO. 12).  
     
     
         6 . The crystalline composition according to claims  2 ,  3  or  4  wherein the ITK kinase domain is ITK/KD/G354 (SEQ ID NO. 1).  
     
     
         7 . The crystalline composition according to  claim 1  wherein the crystals have a hexagonal unit cell whereby unit cell parameters are limited to a=b≠c and alpha=beta=90° and gamma=120°, and the crystal space group symmetry is P6 4  and has unit cell dimensions a=b=239.68 Å, c=97.12 Å wherein the unit cell parameters can vary by 1 to 2%.  
     
     
         8 . The crystalline composition according to any of claims  1 - 7 , wherein said ITK kinase domain-ligand complex has a three-dimensional structure comprising the atomic coordinates as defined in Table 9.  
     
     
         9 . The crystalline composition according to any of claims  1 - 7 , wherein the ligand is one chosen from Table 2.  
     
     
         10 . An isolated polypeptide binding pocket comprising the homologous amino acid residues, based on the kinase-domain residue alignments presented in Table 10, to Q367, I369, L379, K387, and F437 of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 0.42 Angstroms from the coordinates given in Table 9.  
     
     
         11 . An isolated polypeptide binding pocket wherein the isolated polypeptide binding pocket comprises amino acid residues Q367, I369, L379, K387, and F437 of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 3 Angstroms from the coordinates given in Table 9.  
     
     
         12 . An isolated polypeptide binding pocket comprising the homologous amino acid residues, based on the kinase-domain residue alignments presented in Table 10, to V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489, G370, S371, E406, M410, S499, D500, F501, Q367, I369, L379, K387 (RES-SET1) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 0.79 Angstroms from the coordinates given in Table 9.  
     
     
         13 . An isolated polypeptide binding pocket wherein the isolated polypeptide binding pocket comprises amino acid residues V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489, G370, S371, E406, M410, S499, D500, F501, Q367, I369, L379, K387 (RES-SET1) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 3 Angstroms from the coordinates given in Table 9.  
     
     
         14 . An isolated polypeptide binding pocket comprising the homologous amino acid residues, based on the kinase-domain residue alignments presented in Table 10, to V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489, G370, S371, E406, M410, S499, D500, F501 (RES-SET2) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 0.74 Angstroms from the coordinates given in Table 9.  
     
     
         15 . An isolated polypeptide binding pocket wherein the isolated polypeptide binding pocket comprises amino acid residues V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489, G370, S371, E406, M410, S499, D500, F501 (RES-SET2) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 3 Angstroms from the coordinates given in Table 9.  
     
     
         16 . An isolated polypeptide binding pocket comprising the homologous amino acid residues, based on the kinase-domain residue alignments presented in Table 10, to V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489 (RES-SET3) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 0.60 Angstroms from the coordinates given in Table 9.  
     
     
         17 . An isolated polypeptide binding pocket wherein the isolated polypeptide binding pocket comprises amino acid residues V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489 (RES-SET3) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 3 Angstroms from the coordinates given in Table 9.  
     
     
         18 . An isolated polypeptide binding pocket comprising the homologous amino acid residues, based on the kinase-domain residue alignments presented in Table 10, to V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489, Q367, I369, L379, K387 (RES-SET4) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 0.47 Angstroms from the coordinates given in Table 9.  
     
     
         19 . An isolated polypeptide binding pocket wherein the isolated polypeptide binding pocket comprises amino acid residues V377, A389, K391, V419, L433, F435, E436, F437, M438, E439, H440, G441, C442, R486, L489, Q367, I369, L379, K387 (RES-SET4) of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 3 Angstroms from the coordinates given in Table 9.  
     
     
         20 . The isolated polypeptide binding pocket according to any one of claims  10 - 19  wherein the ligand is one chosen from Table 2.  
     
     
         21 . The binding pocket according to  claim 10 , wherein F437 of said binding pocket interacts with a ligand, said interaction resulting in a distance of about 3.8 Angstroms between F437 and the ligand.  
     
     
         22 . A method of obtaining crystals of the ITK kinase domain protein of SEQ ID NO. 1 in a complex with a ligand, said process comprising: 
 (a) obtaining a crystallizable composition, with said crystallizable composition comprising an ITK kinase domain protein, cations and a ligand; and    (b) subjecting the composition of step (a) to conditions which promote crystallization.    
     
     
         23 . The process according to  claim 22 , wherein the ligand is one chosen from Table 2.  
     
     
         24 . A method of identifying an ITK inhibitor, said method comprising: 
 identifying a compound as a potential inhibitor by performing rational drug design with a three-dimensional structure determined for the crystalline composition according to  claim 1;     synthesizing the compound;    determining whether the compound inhibits the activity of ITK.    
     
     
         25 . The method according to  claim 24 , wherein the rational drug design is performed in conjunction with computer modeling.  
     
     
         26 . A method of identifying an ITK inhibitor, said method comprising: 
 contacting a compound with a binding pocket according to  claim 10;     determining whether the compound inhibits the activity of ITK.    
     
     
         27 . A computer assisted method for identifying an inhibitor of ITK activity comprising: 
 supplying a computer modeling application with a set of coordinates of the binding pocket according to any one of claims  10 - 19 ;    supplying a computer modeling application with a set of coordinates of one or more chemical entities;    scoring the binding modes for said one or more chemical entities; and    selecting an inhibitor based on the assigned binding mode scores.    
     
     
         28 . The method according to  claim 27  wherein the computer modeling application is further supplied with a set of coordinates in Table 9.  
     
     
         29 . A method of growing crystals comprising 
 providing a solution of SEQ ID NO. 1 polypeptide complexed with a ligand;    providing a precipitant solution; and    combining the precipitant and solution of SEQ ID NO. 1 polypeptide complexed with a ligand; and    allowing crystals of SEQ ID NO. 1 polypeptide complexed with a ligand to form.    
     
     
         30 . The method according to  claim 29  wherein the precipitant is 5 to 15% polyethylene glycol 1500 (PEG 1500) and is provided by vapor diffusion.  
     
     
         31 . The method according to  claim 29  wherein the precipitant is 8 to 12% polyethylene glycol 1500 (PEG 1500).  
     
     
         32 . An isolated polypeptide chosen from SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 12.  
     
     
         33 . A method of determining the three-dimensional structure of a complex comprising one or more ligands and an ITK kinase domain, comprising: 
 a) crystallizing the ITK kinase domain-ligand complex;    b) obtaining X-ray diffraction data for the crystals of said ITK kinase domain-ligand complex;    c) using the atomic coordinates of Table 9 to determine the three dimensional structure of the ITK kinase domain-ligand complex.    
     
     
         34 . An isolated polypeptide chosen from SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16.  
     
     
         35 . An isolated polypeptide binding pocket comprising amino acid residues F435, M438, and C442 of SEQ ID NO. 1, numbered based on the position in the full-length, wild-type human ITK kinase wherein the backbone-atom R.m.s.d is less than 3 Angstroms from the coordinates given in Table 9.  
     
     
         36 . The binding pocket according to  claim 35 , wherein F435, M438, and C442 of said binding pocket interacts with a ligand, said interaction resulting in a distance of about 4 Angstroms between the ligand and the aromatic side chain of F435, of about 2.7 Angstroms between the ligand and the backbone nitrogen atom of M438, of about 3.1 Angstroms between the ligand and the backbone carbonyl oxygen of M438, and of about 2.7 Angstroms between the ligand and the side chain sulfur atom of C442.  
     
     
         37 . An isolated polypeptide binding pocket comprising amino acid residues F435 and M438 of SEQ ID NO. 1, numbered based on the position in the full length, wild type human ITK kinase, wherein F435 and M438 of said binding pocket interacts with a ligand, said interaction resulting in a distance of about 4 Angstroms between the ligand and the aromatic side chain of F435, of about 2.7 Angstroms between the ligand and the backbone nitrogen atom of M438, and of about 3.1 Angstroms between the ligand and the backbone carbonyl oxygen of M438.